Anti-A, Anti-B o Anti-AB
monoclonal
SUMMARY
In the year 1900 Landsteiner discovered that human red
blood cells could be classified in A, B, AB or O according to
the presence (groups A, B, or AB) or absence (group O) of
highly reactive antigens in its surface. He also demonstrated
that there are antibodies (agglutinins) for A and B antigens
and that one person’s serum does not contain antibodies
for the antigen present in his own red blood cells, but for
the ones he does not have. Nowadays, subgroups of the A
and B groups with different specificity have been identified.
These observations demonstrated the significance of the
ABO compatibility in the transfusion practice. Therefore, ABO
blood grouping typification is the basic test on which all other
pretransfusion tests are based.
PRINCIPLE
The patient red blood cells ar in contact with Anti-A, Anti-B or
Anti-A,B monoclonal antibodies. The presence or absenceof
agglutination of the tested erythrocytes with each reagent indi-
cates the presence or absence of the corresponding antigens.
PROVIDED REAGENTS
Anti-A, Anti-B or Anti-A,B monoclonal: reagents prepared
from monoclonal antibodies IgM class secreted by cellular
lines of mouse hybridomas in a buffered solution containing
< 1 g/l sodium azide as preservative. The involved clones
and each product's color are detailed in the following table:
Product trate, phosphate, dextrose) or CPDA-1 (citrate, phosphate,
Cell lines Color
Name
Anti-A BIRMA-1 Blue
Anti-B LB-2 Yellow
Anti-AB BIRMA-1/ES-4/ES-15 Colorless
The antiserum is characterized by its high potency, avidity
and specificity.
Since they are not from human origin, there is no risk of HIV,
HBV or HCV infection.
NON-PROVIDED REAGENTS
The following may be required, according to the technique
to be used:
- Saline solution
- Phosphate buffered saline solution (PBS) pH 7.0 ± 0.2
- Low ionic strength saline solution (LISS)
INSTRUCTIONS FOR USE
The Reagents are provided ready to use. Do not dilute.
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Reagents for the determination of ABO blood groups
STABILITY AND STORAGE INSTRUCTIONS
The Provided Reagents are stable in refrigerator (2-10oC)
until the expiration date shown on the box. Do not freeze.
Extended storage periods at temperatures outside this range
may increase the reagent reactivity loss.
Avoid repeated thermal changes and regulate the reagent’s
exposure to room temperature to the strictly necessary. In
these usage and storage conditions, the reagent, after being
opened, is stable until the expiration date shown on the box.
INSTABILITY OR DETERIORATION OF REAGENTS
Discard the reagent if contamination is observed. Even
though sodium azide is added as bacteriostatic, it is
recommended to visually inspect the reagent before use.
Do not use in case of turbidity.
The reagent should not be used in the presence of
precipitates or particles.
SAMPLE
Red blood cells or whole blood
a) Collection: blood should be aseptically obtained, with our
without anticoagulant. For the slide technique, blood collected
by digital puncture may be used. To avoid blood clotting when
using this technique, mix with the reagent immediately. For
cord blood tests, red blood cells should be previously washed
with saline solution.
b) Additives: the following may be used as anticoagulant:
EDTA, heparin, ACD (citric acid, citrate, dextrose), CPD (ci-
dextrose, adenine). Wiener lab.’s Anticoagulante W may
be used.
c) Known Interfering Substances: samples with hemolysis
or microbial contamination should not be tested.
d) Stability and storage instructions: samples should be
tested as soon as possible. If the test is not performed im-
mediately, samples should be stored in refrigerator (2-10oC).
In case heparin or EDTA is used for collection, typification
should be performed within 48 hours from collection. Samples
collected with ACD, CPD or CPDA-1 may be tested within
35 days from collection. In case clots are used, typification
should be performed within 7 days from collection. The blood
from donors may be tested until its expiration date.
REQUIRED MATERIAL (non-provided)
- Centrifuge
- Hemolysis tubes
- Slides
- Disposable mixing rods
WARNINGS
Reagents are for “in vitro” diagnostic use and strictly intended
for professional use by qualified personnel with proven im-
munohematology knowledge.
Do not use the reagent after the expiration date.
Azide may react with lead or copper pipes generating
explosive compounds. When discarding the reagent, let run
a copious tab water flow.
Employ the reagents following the ordinary work precautions
used at the clinical chemistry lab.
Reagents and samples should be discarded according to the
local regulations in force.
PROCEDURE
The reagents have been standardized according to the
procedures detailed below. The usage performance with
other techniques is not guaranteed.
In the following techniques the red blood cells suspension
to be tested should be prepared with saline, PBS or LISS.
I- SLIDE TECHNIQUE
Prepare a red blood cells suspension at 10%. A suspen-
sion at 35-45%, in plasma of the same patient or whole
blood, may be used as an alternative.
1) Add 1 drop of red blood cells suspension to 1 drop of
Reagent Anti-A, Anti-B or Anti-A,B monoclonal placed
on a clean and labeled slide. The provided dropper dis-
penses a 50 ± 5 ul volume. The reagent:cell ratio should
be maintained for all assay systems.
2) Mix the Reagent and blood cells with a disposable rod
covering a circular area of 2 cm diameter and continually
balance the slide for 2 minutes.
Observe the macroscopically visible agglutination up to
2 minutes. The observation may be simplified if a diffuse
light source is used. Microscope should not be used.
The test should be interpreted within 2 minutes to
avoid reagent drying due to evaporation. When weak
agglutination is observed, the test should be repeated
using the tube technique (by centrifugation). Two reagent
volumes could be added to 1 sample volume to stress
agglutination, not having the risk of obtaining false
positive results.
II- TUBE TECHNIQUE (by centrifugation)
1) Add 1 drop of red blood cells suspension at 3-5% to
1 drop of Reagent Anti-A, Anti-B or Anti-A,B monoclonal
placed on a hemolysis tube.
2) Mix and centrifuge for 20 seconds at 1,000 g.
3) Stir the tube to detach the cells and examine mac-
roscopically the presence or absence of agglutination.
The observation may be simplified if a diffuse light
source is used.
III- TUBE TECHNIQUE (by sedimentation)
1) Add 1 drop of red blood cells suspension at 3-5% to
1 drop of Reagent Anti-A, Anti-B or Anti-A,B monoclonal
placed on a hemolysis tube.
2) Mix and incubate for 60 minutes at room temperature.
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3) Stir the tube to detach the cells and examine mac-
roscopically the agglutination. The observation may
be simplified if a diffuse light source is used. Read
the tube tests immediately and interpret the results
without delays.
INTERPRETATION OF RESULTS
Slide technique
Positive reaction: the erythrocytes agglutinate within seconds
and stay agglutinated when the plate is balanced. It indicates
the presence of the corresponding erythrocyte antigen.
Negative reaction: if no agglutination is observed after 2
minutes, it indicates the absence of the corresponding
antigen.
Tube technique
Reading: gently tap the tube to detach the sediment and
ma-croscopically examine the presence or absence of
aggluti-nation.
Negative reaction: if no agglutination is observed after 2
minutes, it indicates the absence of the corresponding
antigen. The red blood cell resuspension is homogeneous.
Positive reaction: the erythrocytes agglutinate within seconds
and stay agglutinated after resuspension. It indicates the
presence of the corresponding erythrocyte antigen.
In case of doubt, it is recommended to wait for 2 minutes.
The obtained results in the red blood cell tests (direct),
except the ones using newborn blood samples, should be
confirmed by a serum test (reverse), using A1, A2, B and O
typified ery-throcytes. The expected profiles are shown in
the following table:
Direct test Reverse test
(Red blood cells) (Serum) Group
Anti-A Anti-B Anti-AB Cell A1 Cell A2 Cell B Cell O
- - - + + + - O
+ - + - - + - A
- + + + + - - B
+ + + - - - - AB
Any discrepancy between the direct and reverse tests should
be investigated and determined before reporting the blood
group.
QUALITY CONTROL METHOD
The quality control method should be performed assaying
typified red blood cells. The reagent quality control process
is essential and should be performed at the begining of each
work day with A1, A2, B and O red blood cells.
PROCEDURE LIMITATIONS
Erroneous results may be obserced in the following condi-
tions:
- Test material contamination.
- Incorrect incubation time.
- Recommended incubation time reduction.
- Newborns: the A and B antigens are not fully expressed
 in the newborn. Therefore, it should be handled with care, - Walker Rh, ed. Technical manual. 11th edition. Bethesda:
mostly on premature babies. American Association of Blood Banks, 1993.
- Anti-A and Anti-B reactivity may be reduced and eventually - Garraty G, Postoway N. et al. - Spontaneous agglutination
disappear from serum or plasma of very young patients, of red cells with a positive direct antiglobulin test in various
elder adults or immunosuppressed individuals. media. - Transfusion 24:214-217, 1984.
- Leukemia or other harmful disorders. - Voak D., et al. - Monoclonal anti-A and anti-B development
- Recently transfused patients with important quantities of as cost effective reagents. - Med. Lab. Sci. 39, 109-122,
non-identical blood group may show a mixed field sero- 1982.
logical reaction. - Rouger Ph. et Salmon Ch. La pratique des groupes san-
- False positive reactions: some problems were observed guins et groupes érythrocytaires, 1981.
caused by the typification of the ABO blood groups due - Mollison P.L ,Blood Transfusion - 10ème édition Blackwell
to antibodies that react with drugs, colorants, chemical Science Ed., 1997.
compounds or with colloidal silica particles released by - Moore S. et al. - A Mouse Monoclonal Antibody with Anti-A
bad-quality glasses. (B) Specificity Which Agglutinates AXCells. - Vox Sang,
- Cold agglutinins: in the presence of cold agglutinins, the red Vol. 47, pp. 427-434, 1984.
blood cells washing step performed 4-6 times with saline -Technical Manual of the American Association of Blood
solution, is generally enough to obtain appropriate cells Banks. 10th edition 1990.
for typification. A heating process at 37oC for 10 minutes
before the above mentioned washing is rarely required. A
drop of these cells with 2 drops of saline solution is placed
as control. Agglutination should not be produced.
- Slide tests are not recommended for weak subgroup deter-
mination.
- If the tube technique is used, excessive agitation may dis-
integrate weak agglutinations and produce false negative
results.
- It is important to use the recommended g force during
cen-trifugation, since an excessive centrifugation may
produce difficulties for cell button resuspension and an
insufficient centrifugation may cause easily disintegrating
agglutinations.
- The intensity of some erythrocyte antigen expression may
decrease during storage, especially in clotted samples or
samples collected with EDTA. Optimal results are obtained
with fresh samples.

WIENER LAB. PROVIDES

Anti-A: vial x 10 ml (Cat. Nr. 1443152).
Anti-B: vial x 10 ml (Cat. Nr. 1443154).
Anti-AB: vial x 10 ml (Cat. Nr. 1443153).

REFERENCES
- Wiener AS. - Blood Groups and Transfusion - Charles C.
Thomas 1943.
- Moore, S. et al. - “Int. Symp. on Monoclonal Antibodies:
Standardization of their Characterization and use” - París,
France, 1983. Develop. Biol. Standard 57:49-59.
- Widmann F.K. ed Technical Manual 10th Ed Washington DC,
American Association of Blood Banks 1990, Chapter 11.
- Race R. R and Sanger R. - Blood Groups in Man, 6th Edition
Oxford Blackwell Scientific Publishers 1975:178.
- Issitt P.D. Applied Blood Group Serology 3rd Edition, Montgomery
Scientific Publications, Miami, Florida, USA, 1985, Chapter 10.
- Issit, P.D and Anstee, D.JApplied Blood Group Serology - 4th
Ed. Montgomery Scientific Publications, 1998.
- Pittiglio DH, Baldwin AJ, Sohmer PR. - Modern blood ban-
king and transfusion practices - Philadelphia: FA. Davis,
1987, c.1983.

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Symbols
The following symbols are used in packaging for Wiener lab. diagnostic reagent kits.

C
This product fulfills the requirements of the Euro-
pean Directive 98/79 EC for "in vitro" diagnostic M Manufactured by:
medical devices

P
Xn

Authorized representative in the European Com-

munity Harmful

V "In vitro" diagnostic medical device

Corrosive / Caustic

X Contains sufficient for <n> tests

Xi

Irritant

H Use by

i Consult instructions for use

l Temperature limitation (store at)

 Do not freeze
Calibr. Calibrator

b Control

F Biological risks

Volume after reconstitution

b Positive Control

Cont. Contents c Negative Control

g Batch code h Catalog number

M Wiener Laboratorios S.A.I.C.

Riobamba 2944
2000 - Rosario - Argentina
http://www.wiener-lab.com.ar

Wiener lab.
Dir. Téc.: Viviana E. Cétola
Biochemist
A.N.M.A.T. Registered product
Disp. Nº: 1076/89 (Anti-A)
Disp. Nº: 1073/89 (Anti-B)
Disp. Nº: 1071/89 (Anti-A,B) 2000 Rosario - Argentina
867034000 / 00 p. 4/4 UR110504